During the past project period we have shown that highly purified bovine rod outer segment (ROS) discs contain a 220,000 MW glycoprotein and a number of minor integral membrane polypeptides in addition to rhodopsin. In this renewal application we proposed to extend analysis of these proteins using biochemical, immunological and microscopic techniques to 1) study the distribution, organization and molecular properties of these proteins in bovine ROS disc and plasma membranes and 2) to initiate studies to elucidate their role in visual excitation or other regulatory processes of rod cells. The monoclonal antibody technique will be used with radioimmune assays to prepare and characterize antibodies against specific disc membrane proteins. These antibodies will be used with electron dense visual markers to map the distribution of these proteins along retinal rod cells by scanning electron microscopy (SEM). Their orientation in discs will be determined by transmission electron microscopy (TEM). Interaction between polypeptides in light and dark adapted disc membranes will be studied using chemical crosslinking reagents in conjunction with two-dimensional SDS-gel electrophoresis. Lectin and antibody affinity chromatography, gel filtration and gel electrophoresis will be used to isolate Triton X-100 solubilized disc proteins. The chemical, structural and functional properties of these proteins will be investigated and correlated with their properties in disc membranes. In a related study antibodies against proteolytic fragments of rhodopsin will be used with immunomicroscopic labeling techniques to determine the orientation and accessibility of specific regions of rhodopsin in disc membranes. Finally, magnetic microspheres recently developed in our laboratory will be conjugated to ouabain, lectins and antibodies and used to localize Na ion K ion ATPase and other proteins on photoreceptor cells by SEM and TEM. These novel reagents will also be used to isolate rod cell plasma membranes by magnetic and centrifugation techniques.